Interpretive Summary: Styrax japonicus is a small, low branched tree that produces white flowers. Germination of Styrax seeds was improved by one month of warm stratification (WS; 20°C) followed by 2 to 3 months of cold stratification (CS; 5°C). Gibberellic acid (GA) treatment improved germination only when seeds received a sub-optimal CS. In general, gibberellins break dormancy of seeds which requires CS to promote germination and ABA induces and maintains seed dormancy. Research on the quantification of endogenous GAs and ABA has not been performed in Styrax japonicus seeds. Therefore, this study was initiated to study the water status in seeds using 1H-NMR and to quantify various GAs and also ABA and its precursor and verify the presence of early-13-hydrolylation pathway of GAs in Styrax japonicus seeds as influenced by WS and CS. Styrax seeds germinated well when seeds received 35 days of WS (35D WS) followed by 63 days of CS (63D CS) (35D WS + 63D CS). Seeds that received only 35D WS failed to germinate. Endogenous GA1, GA8, GA19, GA20, and GA53 was identified as well as GA17, GA23, GA28, and GA29. Quantities of GA53 and GA19 increased following WS and WS + CS; however GA1 increased only after WS + CS treatments. The quantity of ABA was decreased by warm treatment but little changed by CS. It was concluded that seed germination was increased by: water content and mobility of water molecules in the cotyledons and endosperm following WS and WS + CS; decreased ABA quantity by WS induced GA biosynthesis leading to GA53 or GA20; and increased concentration of GA1 after CS.

Technical Abstract:
Germination of Styrax japonicus Sieb. et. Zucc. seeds was promoted by warm stratification (WS) at around 20 °C followed by cold stratification (CS) at around 5oC. Biochemical and physiological changes in Styrax seeds during these WS and CS treatments were not investigated. The objective of this work was to analyze the state and mobility of water molecules measured by 1H-NMR, to study the seed surface with ascanning electron microscope, and to determine endogenous gibberellins (GAs) and abscisic acid (ABA) by LC/MS/MS as influenced by WS and CS treatments. Styrax seeds germinated well when seeds received 35 days of WS (35D WS) followed by 63 days of CS (63D CS) (35D WS + 63D CS). Seeds that received only 35D WS failed to germinate. Cotyledon water content increased following 35D WS, but changed little following 35D WS + 63D CS. Following 35D WS + 63D CS, endosperm water content increased and mobility of water to the endosperm increased. Endogenous GA1, GA8, GA19, GA20, and GA53 were identified as well as GA17, GA23, GA28, and GA29 by GC/MS and UPLC/MS/MS which suggested that early-13-hydrolylation pathway [-GA53-(GA44)-GA19-GA20-GA1- GA8] of GAs functioning in the seeds. Quantities of GA53 and GA19 increased following WS and WS + CS; GA1 increased only after WS + CS treatments. Quantity of ABA was drastically decreased by warm treatment but little changed by cold treatment. The effect of WS in Styrax japonicus was not considered to be related to a partial dormancy breaking and the seed coat (testa) may not pose a barrier to water absorption. It was concluded that seed germination was increased by: water content and mobility of water molecules in the cotyledons and endosperm following WS and WS + CS; decreased ABA quantity by WS induced GA biosynthesis leading to GA53 or GA20; and increased concentration of GA1 after CS.